Phosphoprotein target for insulin and its antagonists

ABSTRACT

This invention provides methods for diagnosing and treating individuals with insulin resistance.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/114,540, entitled “PHOSPHOPROTEIN TARGET FOR INSULIN AND ITSANTAGONISTS,” filed on Apr. 1, 2002, which claims the benefit of U.S.provisional Application Ser. No. 60/280,584, filed on Mar. 30, 2001,both of which are incorporated by reference herein.

TECHNICAL FIELD

The invention relates to the field of proteomics. More specifically, itrelates to a phosphoprotein target that exhibits distinctphosphorylation patterns in response to insulin and its antagonists andin certain disease states.

BACKGROUND OF THE INVENTION

Publications referred to by document numbering in this specificationcorrespond to the document list at the end of the specification.

Insulin resistance is characterised by diminished insulin sensitivity oftarget tissues including liver, skeletal muscle and adipocytes (1). Itis a key factor in the pathogenesis of type II diabetes mellitus and isalso associated with other pathological states, such as obesity,dyslipidaemia, hyperinsulinaemia, hypertension and cardiovasculardisease. These clustering metabolic defects have been termed syndrome“X” or “the insulin resistance syndrome” (2).

The molecular basis of insulin resistance is extremely complex andmultifactorial. Defects in several steps of insulin action, such as theactivation of insulin receptors, post-receptor signal transduction andthe glucose transport effector system, have been implicated in thisdisease (3, 4). Defective insulin receptor kinase activity, reducedIRS-1 tyrosine phosphorylation and decreased PI-3 kinase activity wereobserved in both human type II diabetic patients as well as animalmodels such as ob/ob mice (5, 6).

In addition to the intrinsic defects of the insulin receptor andpostreceptor signalling components, other circulating factors, such asTNF-α., leptin, free fatty acids (FFA) and amylin may also contribute tothe pathogenesis of insulin resistance (7-11). For instance, amylin, ahormone co-secreted with insulin from pancreatic islet β-cells, has beenshown to antagonise insulin's metabolic actions both in vivo and invitro (12-16). It can inhibit insulin-stimulated glucose uptake andglycogen synthesis. In vivo administration of amylin resulted inhyperglycemia and induced insulin resistance, similar to that observedin type II diabetes. Although some earlier studies suggested thatamylin's biological effects on fuel metabolism were only ofpharmacological interest, more recent in vivo studies with anamylin-selective antagonist have strongly supported its physiologicalrelevance (17). Moreover, amylin-deficient mice showed increased insulinresponsiveness and more rapid blood glucose elimination followingglucose loading, further confirming the role of amylin in the causationof insulin resistance (18). Indeed, elevated levels of circulatingamylin (hyperamylinemia) and an increased ratio of amylin to insulinwere observed in patients with type II diabetes as well as otherdiseases associated with insulin resistance, such as obesity and glucoseintolerance (19).

Despite these advances, the detailed cellular mechanisms of insulinresistance are far from clear and there is a need for new therapeuticand diagnostic modalities for this condition.

SUMMARY OF THE INVENTION

The invention provides, in one aspect, a method for screening for anagent useful for treatment of insulin resistance by contacting amammalian cell expressing P20 and the agent and determining if the agentsuppresses the level of at least one of P20 isoforms S2 and S3, whereinthe suppression of S2 and S3 levels is indicative of an agent useful fortreatment of insulin resistance. In one embodiment, the mammalian cellis insulin resistant. In another embodiment, the mammalian cell is froma rat or human. In another embodiment, the mammalian cell is a myocyte,adipocyte, or skeletal muscle cell. In another embodiment, the agent iscontacted with isolated skeletal muscle. In another embodiment, thecontacting occurs by administration of the test agent to an animal(e.g., a rodent with genetic or experimentally induced insulinresistance). In another embodiment, the cell is exposed to an amount ofamylin, CGRP1, CGRP2, epinephrine or norepinephrine sufficient to inducephosphorylation of P20 during, prior to, or after contacting the celland the test agent. In another embodiment, the cell is exposed to anamount of insulin sufficient to reduce amylin-induced phosphorylation ofP20 in a non-insulin resistant cell, during, prior to, or aftercontacting the cell and the test agent. In another embodiment, the cellis exposed to insulin ex vivo.

In another aspect, the invention provides a method for screening for anagent useful for treatment of insulin resistance by: (a) contacting aninsulin resistant mammalian cell expressing P20 and the agent; (b)determining an expression level of at least one of P20 isoforms S2 andS3 in the cell; and (c) comparing the expression level of S2 and/or S3to a reference expression level of S2 or S3, wherein said referenceexpression level is characteristic of (i) expression in a similar cellnot exposed to the agent or (ii) expression in a cell that is notinsulin resistant, and wherein an expression level that is lower than(i) or similar to (ii) indicates the agent is useful for treatment ofinsulin resistance. In one embodiment, the mammalian cell is from a rator human. In another embodiment, the mammalian cell is a myocyte,adipocyte, or skeletal muscle cell. In another embodiment, the agent iscontacted with isolated skeletal muscle. In another embodiment, thecontacting occurs by administration of the test agent to an animal(e.g., rodent with genetic or experimentally induced insulin resistance)

In another aspect, the invention provides a method for diagnosinginsulin resistance in an individual by obtaining a biological samplefrom the individual and determining a level of at least one of P20isoforms S2 and S3, wherein the individual is diagnosed as being insulinresistant when the level of expression of at least one of S2 and S3 ishigher than a reference level characteristic of an individual notsuffering from insulin resistance. In one embodiment, the cells in thebiological sample are contacted with insulin ex vivo. In anotherembodiment, the levels of both S2 and S3 are determined. In anotherembodiment, the levels of both S2 and S3 are higher than a referencelevel characteristic of an individual not suffering from insulinresistance.

In another aspect, the invention provides a method of treating insulinresistance in an individual comprising administering a treatment or anagent that reduces the level of P20 isoforms S2 and S3 in theindividual. In one embodiment, the agent is identified by the methods ofscreening described above.

In another aspect, the invention provides the use of an agent thatreduces the level of at least one of S2 and S3 in a cell in thepreparation of a medicament for treatment of insulin resistance.

In another aspect, the invention provides a method of assessing theefficacy of a treatment for insulin resistance in an individual bymonitoring the level of at least one of S2 and S3 in the individual towhom the treatment has been administered.

In another aspect, the invention provides a method for diagnosinginsulin resistance or a propensity to insulin resistance in anindividual by determining the level of expression of at least one of P20isoforms S2 and S3 in a cell of an individual, and comparing the levelto a reference level characteristic of a cell of the same type in anindividual not suffering from insulin resistance or diabetes wherein alevel of expression that is higher than the reference level isdiagnostic of insulin resistance or a propensity to insulin resistancein the individual. In one embodiment, the levels of both S2 and S3 aredetermined. In another embodiment, the levels of both S2 and S3 arehigher than the reference level. In another embodiment, the level ofexpression of S2 and/or S3 is the same as or greater than a secondreference level, wherein said second reference level is characteristicof an individual with insulin resistance.

In another aspect, the invention provides a method of assessing theefficacy of a treatment for insulin resistance in an individual bymonitoring the level of at least one of S2 and S3 in the individual towhom the treatment has been administered.

In another aspect, the invention provides a method of treating insulinresistance in an individual by administering a treatment or an agentthat reduces the level of P20 isoforms S2 and S3 in the individual. Inone embodiment, the agent is identified by the methods of screeningdescribed above.

In another aspect, the invention provides the use of an agent thatreduces the level of at least one of S2 and S3 in a cell in thepreparation of a medicament for treatment of insulin resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows immunoblotting analysis of P20 expression in severaldifferent rat tissues. 50 μg of protein from each of the indicated rattissues was separated by 12.50/% SDS-PAGE, and immunoblotted to detectP20 as described in the Examples. The result is the typicalrepresentation of three independent observations.

FIG. 2 shows a two-dimensional phosphoprotein map of insulin-stimulatedrat extensor digitorum longus (“EDL”) muscle. EDL muscle strips wereradiolabelled with ³²P, treated with 50 nM insulin for 30 minutes, then100 μg protein from each sample was separated by two-dimensionalelectrophoresis and detected by autoradiography. The denoted proteinswere identified either by amino acid sequencing or by western blotanalysis. Note that P20(S1) refers to the isoform of P20 with pI valueof 6.0. The experiments were performed four times and the figure shownis from one representative experiment.

FIG. 3 shows interplay between insulin and its antagonists on thephosphorylation of P20. ³²P-labelled EDL muscle strips were treatedwithout or with different hormones for 30 min at the followingconcentrations: insulin, 50 nM; amylin, 50 nM; epinephrine, 50 nM; andcalcitonin gene-related peptide (“CGRP”), 50 nM. Phosphorylation of P20was analysed by two-dimensional gel electrophoresis (“2-DE”) andquantitated using phosphorimaging software. The table in the lower panelrepresents the quantitative data for the three phosphoisoforms of P20.The results are expressed as mean photostimulated luminescence (“PSL”)values±S.D. for four independent observations. † shows significantdifference (p<0.05) between insulin treated samples and insulin plusamylin treated samples. ‡ shows significant difference (p<0.05) betweenamylin treated samples and insulin plus amylin treated samples. Notethat similar results were observed when adding these agonistssequentially (ie., pre-incubation with insulin for 15 min, followed byaddition of amylin, CGRP or epinephrine for another 15 min, or viceversa).

FIG. 4 shows changes in the phosphorylation of P20 and itsresponsiveness to insulin and amylin in dexamethasone-treated rats withinsulin resistance. EDL muscle strips from non-diabetic control rats(left panel) or rats with insulin resistance (right panel) wereradiolabelled with ³²P, treated with buffer only (A and B); 50 nMinsulin (C and D); 50 nM amylin (E and F); or 50 nM insulin plus 50 nMamylin (G and h) for 30 min. Phosphorylation of P20 was analysed by 2-DEand phosphorimaging. The result is the typical representation of fourindependent observations.

FIG. 5 shows enhanced phosphorylation of S2 and S3 is associated withinsulin resistant rats induced by high-fat feeding. 100 μg of proteinsfrom muscle strips from healthy rats or high fat-induced diabetic ratswere separated by 2-DE and the three phospho-isoforms of P20 (S1, S2 andS3) was visualised by probing with anti-p20 antibody as described inFIG. 1. The table in the lower panel represents the quantitativeanalysis for the abundance of each phospho-isoform of P20 innon-diabetic control rats and high fat induced diabetic rats. Theabundance of each isoform is expressed as mean PSL values±S.D. *indicates the values that are significantly different (P<0.01) fromcorresponding values in control rats (n=4).

FIG. 6 shows mRNA abundance and protein concentration of P20 is notaltered in rats treated with dexamethasone. Panel I: northern blotanalysis. RNA was prepared from the EDL muscles of saline (A) ordexamethasone-injected rats (B), and also from the EDL muscle stripstreated without (C) or with 50 nM amylin (D) for 30 min in vitro,blotted and probed with the labelled P20 cDNA. The negative image of theethidium bromide-stained RNA loaded in each lane is also shown.Quantitative analysis was performed using a phosphorimager. Panel II:western blot analysis of P20. 30 μg of total proteins from EDL musclestreated as in panel I was separated by 12.5% SDS-PAGE, probed with antiP20 antibody as in FIG. 1. The table in Panel III represents theincreased/decreased fold in P20 mRNA and protein level under therespective treatment, relative to saline-treated control rats. Theresult is expressed as the mean±S.D. from three individual experiments.

FIG. 7 shows the effect of P20 over-expression on glucose uptake in L6myotubes. A: L6 cells were transfected with pCXN2-GLUT4myc, orpCXN2-GLUT4myc and pcDNA.P20. Following selection with 400 μg/ml G418,clones expressing myc-tagged GLUT4 alone (GLUT4myc) and clonesexpressing both myc-tagged GLUT4 and P20 (GLUT4myc+P20) were expanded,and differentiated as described in the Methods. 30 μg of cell lysatesfrom L6 myotubes were separated by 10% SDS-PAGE. The levels of P20 andmyc-tagged GLUT4 expression were analysed by western blot, usingspecific anti-p20 and anti-GLUT4 antibodies respectively. B: The celllines selected in A were differentiated in 6-well plates, and assayedfor 2-deoxyglucose uptake in response to insulin or insulin plus amylinas described in the Methods (n=4, expressed as mean±S.D.). Note that theFigure shows the result of a typical experiment, and that similarresults were also obtained from at least another two independenttransfectants which express myc-tagged GLUT4, or myc-tagged GLUT4 plusP20. * indicates the values that are significantly different (P<0.01)from corresponding values in cells overexpressing GLUT4myc alone.

DETAILED DESCRIPTION OF THE INVENTION

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,nucleic acid chemistry, and immunology, which are within the skill ofthe art. Such techniques are explained fully in the literature, such as,Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.,1989) and Molecular Cloning: A Laboratory Manual, third edition(Sambrook and Russel, 2001), jointly referred to herein as “Sambrook”);Current Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987, including supplements through 2001); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, and Harlow and Lane (1999) Using Antibodies: A Laboratory ManualCold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. jointlyreferred to herein as “Harlow and Lane”), Beaucage et al. eds., CurrentProtocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York,2000).

II. Introduction

We have recently used comparative proteomic analysis to systematicallyinvestigate the phosphorylation cascades evoked by insulin and itsantagonists in rat skeletal muscle, and have identified a novelphosphoprotein P20 as the common intracellular target of these hormones(23, 24). Insulin and its antagonistic hormones amylin, epinephrine andcalcitonin gene-related peptide (CGRP), through distinctive signalingpathways, phosphorylate P20 at different serine residues to producemultiple phospho-isoforms of this protein. In the Examples, infra, wedemonstrate that P20 in skeletal muscle from diabetic rats with insulinresistance has an abnormal phosphorylation pattern, although theexpression level of this protein is not changed. Moreover, theresponsiveness of P20 to insulin and amylin is also altered in insulinresistant animals.

Our results demonstrate that insulin resistance in skeletal muscle isassociated with the appearance of the two P20 phospho-isoforms S2 andS3, and also with the inability of insulin to suppress theamylin-mediated phosphorylation of these two isoforms. Thus, increasedphosphorylation of two isoforms of P20, S2 and S3, is associated withinsulin resistant states in general. In the absence of hormonestimulation, phosphorylation of S2 and S3 is hardly detected in thenon-diabetic cells and animals, e.g., in muscle samples, but twophospho-isoforms are abundant in cells from the insulin resistantanimals (e.g., about 5-fold higher than non-insulin resistant animals).

Further, in insulin resistant cells, addition of insulin decreasedamylin-induced phosphorylation of S2 and S3 is greatly attenuatedcompared to normal (non insulin resistant) cells or animals.

As used herein, “P20” means the protein cloned by Inayuma, Y. et al Gene178(1-2):145-50 (1996) and its homologs in mammalian species. See alsoWang et al., 1999, FEBS Lett 457:49-52, and Wang et al., 1999, FEBS Lett462:25-30, 1999. P20 exists in three phosphorylated isoelectricvariants, referred to as S1, S2 and S3. In rats, S1 (pI=6.0) isphosphorylated at serine 157; S2 (pI=5.9) is phosphorylated at serine16, and S3 (pI=5.6) is phosphorylated at multiple sites, includingserine 16. Homologues of S2 and S3 have been identified in humanskeletal muscle by Western blotting and two dimensional electrophoresisequivalent to the methods employed to identify these phosphoisoforms inrodent tissues. Homologues in other mammals can be characterized usingmethods described herein.

III. Drug Screening Methods

Phosphoprotein P20 and its isoforms or isovariants (e.g., phosphoisoformS1, S2, or S3) can be targets for drug screening purposes. Accordingly,methods of screening compounds that are potential drugs are provided(e.g., methods for screening for an agent useful for treatment ofinsulin resistance or syndrome X-associated conditions). In oneembodiment, the methods of identifying compounds that are potentialdrugs that interact with and/or bind to a phosphoprotein P20 and itsisoforms (e.g., S1, S2 and/or S3 isoforms) are provided. In a relatedaspect, the invention provides methods of screening for drugs whichinteract with and/or bind to a protein or receptor associated with P20or its isoforms (e.g., glucose transporters) are provided.

Preliminary screens can be conducted by screening for compounds capableof binding to P20, as at least some of the compounds so identified arelikely modulators of P20 phosphorylation at the S2 and S3 sites. Bindingcan be detected using standard techniques, such as assays including, butnot limited to, methods that measure co-precipitation, co-migration onnon-denaturing SDS-polyacrylamide gels, and co-migration on Westernblots. The P20 protein utilized in such assays can be naturallyexpressed, cloned or synthesized. The binding can be assessed usingpurified P20, cell-free systems, or intact cells (e.g., recombinantcells expressing P20). In assays, the P20 and/or putative drugs may belabeled with a detectable marker (e.g., radiolabel or a non-isotopiclabel such as biotin or fluorescent marker). Drug candidates can beidentified by choosing compounds which bind with affinity, preferablyhigh affinity, to the phosphoprotein P20 and its isoforms expressed inthe cell, using techniques well known in the art. Drug candidates canalso be screened for selectivity by identifying compounds that bind tophosphoprotein P20 and its isoforms but do not bind to any otherreceptors or receptor sites. In another embodiment, drug candidates arescreened to identify compounds that bind to a protein associated withP20 or its isoforms (e.g., glucose transporter or receptor) and exertits effects. Accordingly, a method of drug screening involves exposingmammalian cells expressing P20 or its isoforms to one or a plurality ofdrugs, then determining those drugs which bind to the phosphoprotein P20and its isoforms expressed in the mammalian cell, and therebyidentifying drugs which interact with and/or bind to the phosphoproteinP20 and its isoforms. Compounds that bind to P20 or P20-associatedproteins can be subjected to additional assays to determine theirtherapeutic activity. Preferred compounds are those that bind P20 andmodulate phosphorylation of S2 and/or S3.

One method that can be used for drug screening involves using mammaliancell(s) that express P20 (or its isoforms), contacting the cells withone or more test compounds, and monitoring the effect of the compound(s)on the cells. One such effect that can be monitored or measured is theuptake of glucose or a variant of glucose (e.g., uptake of2-deoxyglucose, as shown in FIG. 7). Another effect that can bemonitored is the phosphorylation of P20 and/or the generation ofisoforms such as S1, S2, and/or S3. Phosphorylation patterns can beassessed by methods known in the art and by those assays describedherein.

In one aspect, the invention provides a method for identifying an agentuseful for treatment of insulin resistance by contacting a mammaliancell that expresses P20 with a test agent and determining if the agentsuppresses the level of at least one of P20 isoforms S2 and S3 (e.g.,compared to expression in the absence of the test agent). Suppression oflevels of S2 and/or S3 by an agent is indicative that the agent isuseful for treatment of insulin resistance and related conditions (e.g.,syndrome X-related). Suppression means a lower or reduced level of S2and/or S3 compared to a cell of the same cell type not contacted withthe test agent. Preferably a test compound useful for treatment ofinsulin resistance is one that reduces the levels of S2 and/or S3 by atleast about 20%, often by at least about 40%, very often by at leastabout 50%, and sometimes by at least about 60% compared to a controlcell.

In certain embodiments, the mammalian cell is rodent (e.g., rat, mouse,hamster or the like) or primate (e.g., human or non-human primate). Themammalian cell can be an isolated cell or cells (e.g., in in vitro cellculture), a cell in a tissue (e.g., a biopsy tissue), a cell in a testanimal or any other cell. For example, the cell can be a myocyte, amuscle cell (e.g., skeletal muscle, soleus muscle, extensor digitorumlongus muscle, heart muscle, or smooth muscle), an adipocyte, or a bloodcell. Thus, for example, isolated tissues, such as isolated skeletalmuscle tissue can be used (e.g., as is described in the Examples). Anexample of a suitable mammalian cell type is L6 cells, as used in theexperiment depicted by FIG. 7.

Cells expressing P20 can be cells that naturally express this protein.Alternatively, they can be recombinant cells. Phosphoprotein P20 (e.g.,P20 isoforms) can be expressed in mammalian cells by using a plasmid orexpression vector which comprises a genetic sequence (e.g., DNAsequence) which encodes for phosphoprotein P20 (e.g., P20 isoforms).

In an embodiment, the cell is an insulin resistant cell. By “insulinresistant” is meant a cell that demonstrates a sub-normal dose-responsewhen treated with insulin, in an insulin-responsive process or pathwayor, for example, in the activation or inhibition of aninsulin-responsive enzyme and/or is a cell or tissue isolated from ananimal that is insulin resistant. Animals that are insulin resistantinclude, but are not limited to, a human diagnosed with insulinresistance or type II diabetes, animals that are genetically insulinresistant (e.g., ob/ob mice), or animals in which insulin resistance ordiabetes has been experimentally induced (e.g., by administration ofdexamethasone, maintenance on a high fat diet, etc.).

Insulin resistance of ex vivo cultured cells or tissues can be generatedby treatment with amylin, but this is not the only way to achieve suchpreparations. Other molecules that can generate insulin resistancefollowing in vitro treatment of cells or tissues with them, includeCGRP1 or CGRP2; epinephrine; or norepinephrine. In addition,insulin-resistant cells or tissues may be generated by first treating ananimal, such as a rodent, with other hormones that are capable ofgenerating insulin resistance only in vivo and not directly in vitro.Examples in this second category of hormones include glucocorticoidagonists (e.g., cortisol, corticosterone, prednisone or dexamethasone)and other hormones (e.g., growth hormone and growth hormone agonists);hormones in both these classes can evoke insulin resistance in vivo. Exvivo cultures of cells or tissues, such as liver, adipose tissue,skeletal muscle or cardiac muscle, are then prepared from animals madeinsulin resistant by treatment with these hormones, and are employed inthe assays. Cells or tissues generated from animals with geneticallybased insulin resistance and obesity can also be employed in assays.Examples of useful rodent strains are: ob/ob mice, db/db mice, fa/farats and LAN-cp rats. Further sources of cells or tissues that can beusefully employed in such assays are those derived from animals madeinsulin resistant by nutritional manipulations or from insulin resistanthumans. Examples of useful nutritional manipulations for rodents includefeeding to otherwise normal rodents of diets that containsupraphysiological amounts of fat or infraphysiological amounts ofprotein. Insulin resistant cells or cell lines also can be obtained fromthe American Type Culture Collection (ATCC, P.O. Box 1549 Manassas, Va.20108). Insulin resistance of a cell (line) or animal can be determinedeither in vitro or in vivo using routine methods. For example, in vitrotesting can involve incubating mammalian cells with and without insulinand then determining the effect of insulin on glycogen synthesis and/orglucose uptake. In vivo testing can involve administering insulin to amammal in a fasting glucose test and then measuring glycogen synthesisand/or glucose uptake. In humans, insulin resistance can be assessed byany of a variety of methods known in the art (see, e.g., Bergman et al.,1985, Endocrine Review 6:45-86; Reaven et al., 1979, Diabetologia16:17-24).

As noted, suppression of levels of S2 and/or S3 by an agent isindicative that the agent useful for treatment of insulin resistance andrelated conditions (e.g., syndrome X-related conditions). Levels(sometimes referred to as “expression levels”) of S2 and/or S3 in a cellcan be determined by any number of methods including, but not limitedto, two-dimensional gel electrophoresis, chromatographic methods (e.g.,HPLC), immunological methods (e.g., immunoprecipitation using antibodiesspecific for S2 or S3, radioimmune assays (RIA), Western blotting,etc.), and the like, including use of various imaging and analyticalmethods for quantification of levels of specific proteins (e.g.,specific phosphorylated proteins). Conveniently, phosphorylation of P20in cells can be monitored using radioisotopes of phosphorous, forexample as described in the Examples, infra.

The invention further provides a method for screening for an agentuseful for treatment of insulin resistance by contacting a mammaliancell expressing P20 and an agent, determining an expression level of atleast one of P20 isoforms S2 and S3; and comparing the level of at leastone of P20 isoforms S2 and S3 to a reference level. In embodiments, thereference expression level is characteristic of (i) expression in asimilar cell not exposed to the agent or (ii) expression in a cell thatis not insulin resistant. An agent is potentially useful for treatmentof insulin resistance when the expression level in the presence of theagent is lower than (i) or similar to (ii). In this context, “lowerthan” means an expression level of S2 and/or S3 at least about 20%,lower than (i), often at least about 40%, often at least about 50%, andsometimes at least about 60%. In this context, “similar” means anexpression level that is within 2-fold of (ii), preferably within1.5-fold of (ii).

As discussed in the Examples, amylin and insulin have countervailingeffects on the levels of S2 and S3. Amylin (as well as agents such asCGRP1, CGRP2, epinephrine or norepinephrine) can be used to inducephosphorylation of P20 during, prior to, or after contacting the celland the test agent. Insulin (or other insulin agonist) can be contactedwith the cells to measure the insulin dose-response of one or moreprocesses in the tissue (and hence the insulin responsiveness of thecell or tissue). Thus, treatment with an insulin agonist and measurementof an indicator variable is the probe capable of demonstrating insulinresistance in the cell or tissue.

Insulin resistance in skeletal muscle is associated with the appearanceof the two P20 phospho-isoforms S2 and S3, and with the inability ofinsulin to suppress the amylin-mediated phosphorylation of these twoisoforms. In one aspect of the invention, a test agent is assayed forthe ability to restore the ability of insulin to suppress thephosphorylation of S2 and S3.

It will be appreciated that the screening assays of the invention can becarried out in the presence of insulin (or an insulin substitute, suchas an insulin receptor agonist). Thus, in one embodiment, the screeningassay is carried out in the presence of insulin and/or the cell isexposed to insulin or insulin analog at the time of, prior to, or afterthe contacting with the test compound. The insulin can be natural,synthetic, recombinant, primate (e.g., human), or rodent (e.g., rat ormouse). Examples of insulin agonists include, without limitation, anystructure of insulin in which one or more amino acid residues aresubstituted to yield an altered molecule with insulin-like activity(e.g., insulin-like dose-response relationships in vivo or in vitro).Examples of insulin agonists that can be employed in such assaysinclude: human insulin; [LysPro]human insulin (a synthetic analog ofhuman insulin), and rat insulin I or rat insulin II, which are naturallyoccurring homologues of human insulin. The amount of insulin used isusually within the range 1 pM to 1 μM, often 30 nM to 100 nM (e.g., 50nM), i.e., a range that spans the concentration-response of a tissueprocess or pathway that is informative concerning the relative insulinsensitivity of the tissue. Examples of such processes include glucosetransport or incorporation of glucose into glycogen, which areinformative in skeletal muscle; or suppression of basal orglucagon-stimulated glucose output from hepatocytes or the isolatedperfused liver, which inform on liver function. In insulin resistant ordiabetic animals or tissues, amylin-evoked phosphorylation of S2 and S3is not greatly decreased by the administration of insulin, while innormal animals or tissues, insulin significantly decreasesphosphorylation of S2 and S3 (e.g., typically by at least about 30%,more often by at least about 50%). The contacting of cells and insulincan be in vivo or in vitro.

In another embodiment, at the time of, prior to, or after the contactingwith the test compound, the cells (e.g., tissues) used in the screeningassay are exposed to an agent that induces phosphorylation of S2 and/orS3. Exemplary agents are hormones such as amylin, CGRP1, CGRP2,epinephrine or norepinephrine (including analogs of each). The hormone,e.g., amylin, can be natural, synthetic, recombinant, primate (e.g.,human), or rodent (e.g., rat or mouse). The amount of hormoneadministered is an amount sufficient to induce insulin resistance in aninformative pathway or process, such as glucose transport orincorporation of glucose into glycogen, which are informative inskeletal muscle, for example (for amylin) about 10 nM to 100 nM (e.g.,50 nM). The contacting of cells and hormone can be in vivo or in vitro.

Compounds or agents which are contemplated as potential drugs include,but are not limited to, antibodies (polyclonal, monoclonal, recombinant,chimeric, etc.), synthetic molecules, small molecules (e.g., smallorganic molecules), peptides, compounds comprised of nucleic acids, andproteins. One source of potential drugs are libraries of natural orsynthetic compounds. The creation and simultaneous screening of largelibraries of synthetic molecules can be carried out using well-knowntechniques in combinatorial chemistry, for example, see van Breemen(1997) Anal Chem 69:2159-64; Lam (1997) Anticancer Drug Des 12:145167(1997); Gold (1995) J. Biol. Chem. 270:13581-13584). In addition, alarge number of potentially useful activity-modifying compounds can bescreened in extracts from natural products as a source material. Sourcesof such extracts can be from a large number of species of fimgi,actinomyces, algae, insects, protozoa, plants, and bacteria. Thoseextracts showing activity can then be analyzed to isolate the activemolecule. See for example, Turner (1996) J. Ethnopharmacol 51(13):3943;Suh (1995) Anticancer Res. 15:233239. Several methods of automatingassays have been developed in recent years so as to permit screening oftens of thousands of compounds in a short period. See, e.g., Fodor etal., 1991, Science 251:767-73, and other descriptions of chemicaldiversity libraries, which describe means for testing of bindingaffinity by a plurality of compounds.

IV. Diagnostic Methods

The invention provides a method for diagnosing insulin resistance in anindividual by determining the level of expression of at least one of P20isoforms S2 and S3 in a cell of an individual, and comparing the levelto a reference level characteristic of a cell of the same type of anindividual or population of individuals (i) not suffering from insulinresistance or diabetes or (ii) diagnosed with insulin resistance ordiabetes. As used herein, the term “individual” includes mammals such ashumans, non-human primates, commercially valuable animals, pets, andexperimental animals (e.g., rodents including mice and rats).Conveniently the method can be carried out by obtaining a biologicalsample from the individual containing at least one, and preferably many,P20-expressing cells. Examples of such cells include myocytes, musclecells (e.g., skeletal muscle, soleus muscle, extensor digitorum longusmuscle, heart muscle, or smooth muscle), blood cells, and adipocytes.Biological samples can be in the form of tissues (including tissuesobtained by biopsy) or tissue cultures or cells derived therefrom, andthe progeny thereof, cells from blood, whole cells, cell fractions, cellextracts, and cultured cells or cell lines), body fluids (e.g., urine,sputum, amniotic fluid, synovial fluid), or from media (from culturedcells or cell lines), and the like. Biological samples also includecells manipulated after removal from the individual, e.g., by exposureto insulin, amylin, CGRP or epinephrine, or enrichment for specific celltypes (e.g., myocytes or adipocytes).

In one embodiment, the reference level is a level of expressioncharacteristic of a cell of the same type in an individual or populationof individuals not suffering from insulin resistance or diabetes. Inanother embodiment the reference level is a level of expressioncharacteristic of a cell of the same type in an individual or populationof individuals diagnosed with insulin resistance or diabetes. In oneembodiment, either one of S2 and S3 levels are determined. In anotherembodiment, the levels of both S2 and S3 are determined. In oneembodiment, a diagnosis of insulin resistance is made when the levels ofS2 and/or S3 are higher (e.g., statistically significantly higher) thanthe level characteristic of an individual not suffering from insulinresistance or diabetes and/or lower (e.g., statistically significantlylower) than the level characteristic of an individual diagnosed withinsulin resistance or diabetes.

In some cases, it will be desirable to establish normal or baselinevalues (or ranges) for S2 and/or S3 levels. Normal (e.g., low) levelscan be determined for any particular population, subpopulation, or groupof organisms according to standard methods well known to those of skillin the art. Generally, baseline (normal) levels of S2 and/or S3 forhealthy individuals are determined by quantitating the levels inbiological samples obtained from normal (healthy) subjects not sufferingfrom insulin resistance or diabetes. For certain samples and purposes,one may desire to quantitate the amount of S2 and/or S3 with referenceto the total amount of P20 protein in the sample, and/or on a per cellbasis. To determine the cellularity of a sample, one may measure thelevel of a constitutively expressed gene product or other gene productexpressed at known levels in cells of the type from which the sample wastaken. It is possible that normal (baseline) values may differ somewhatbetween different cell types or according to the age, sex, or physicalcondition (other than presence of insulin resistance) of a patient.Thus, for example, when an assay is used to determine changes in S2and/or S3 levels associated with insulin resistance, the cells used todetermine the normal range of expression can be cells from persons ofthe same or a different age, depending on the nature of the inquiry.Application of standard statistical methods permits determination ofbaseline levels of expression, as well as permits identification ofsignificant deviations from such baseline levels. It will be appreciatedthat the assay methods do not necessarily require measurement ofabsolute values of S2 and/or S3, unless it is so desired, becauserelative values are sufficient for many applications of the methods ofthe present invention.

In a different embodiment, the invention provides a method of assessingthe efficacy of a treatment for insulin resistance. The assays of theinvention may also be used to evaluate the efficacy of a particulartherapeutic treatment regime in animal studies, in clinical trials, orin monitoring the treatment of an individual patient. In these cases, itmay be desirable to establish the baseline for the patient prior tocommencing therapy and to repeat the assays one or more times throughthe course of treatment, usually on a regular basis, to evaluate whetherS2 and/or S3 levels are moving toward the desired endpoint (e.g.,reduced expression of S2 and/or S3) as a result of the treatment.

V. Treatment Methods

Without intending to be bound by any particular mechanism, as noted inthe Examples, infra, increased phosphorylation of S2 and S3characteristic of insulin resistance is not due to the increasedexpression of P20, but likely due to a defect in the intracellularsignal transduction pathways that lead to generation of itsphosphorylated isoforms. These results suggest that alterations inphosphorylation of P20 contribute to the development of insulinresistance. Compositions and therapies that reduce the levels of P20isoforms S2 and/or S2 in an individual are thus useful for the treatmentof insulin resistance and related conditions. In accordance with this,the invention provides methods for treating insulin resistance inindividuals by administering a treatment (e.g., compound) that reducesthe level of P20 isoforms S2 and/or S3 in at least one cell in theindividual. As used herein, “treatment” is an approach for obtainingbeneficial or desired results including and preferably clinical results.The beneficial or clinical results include but are not limited to animprovement in an individual's ability to be sensitized to insulin and adecrease in an individual's insulin resistance. A treatment plan mayoccur over a period of time and may involve multiple dosages, multipleadministrations, and/or different routes of administration of atherapeutic agent.

In one embodiment, the agent is identified by the methods of screeningdisclosed herein. The treatment or agent can be administered as apharmaceutical composition. The pharmaceutical composition can include adrug identified by the method described above and a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical compositionsof the invention are formulated for administration by injection (e.g.,intraperitoneally, intravenously, subcutaneously, intramuscularly,etc.). As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.Excipients as well as formulations for parenteral and nonparenteral drugdelivery are set forth in Remington's Pharmaceutical Sciences 19th Ed.Mack Publishing (1995). Once the candidate drug has been shown to beadequately bio-available following a particular route of administration,for example orally or by injection and has been shown to be non-toxicand therapeutically effective in appropriate disease models, the drugmay be administered to patients by that route of administrationdetermined to make the drug bio-available, in an appropriate solid orsolution formulation, to gain the desired therapeutic benefit.

EXAMPLES Example 1

Materials and Methods

Male Wistar rats were fed standard rat chow (NRM Diet 88, Auckland, NewZealand) with water ad libitum. [³²P]-orthophosphate and[¹⁴C(U)]-D-glucose were purchased from ICN. 2-deoxy-D-[³H] glucose (1mCi/ml) was from NEN and iodine-125 from Amersham pharmacia. Humaninsulin was Actrapid from Novo Nordisk. Rat arnylin and CGRP werepurchased from Bachem (Torrance, Calif.); epinephrine was from DavidBull Laboratories; and dexamethasone from Sigma. The two-dimensional gelelectrophoresis (2-DE) system and reagents were from Pharmacia. Anti-P20polyclonal antibody was a generous gift from Dr. Kanefusa Kato (25).Anti-GLUT4 (H-61) was from Santa Cruz. The enhanced chemiluminescence(ECL) detection system was from Boehringer. The total cellular RNAextraction reagent (TRIZOL®), G418, Lipofectamine Plus reagent andrandom priming labelling kits were from Life Technology. pCXN2-GLUT4myc,which expresses myc-tagged GLUT4 in mammalian cells, was kindly providedby Dr David James (The University of Queensland, Australia).

Establishment of the Dexamethasone- or High Fat-Induced Rat Models withInsulin Resistance

All experimental protocols were approved by the Institutional AnimalEthics Committee. Male Wistar rats were injected with dexamethasone (3.1mg/kg/day, intraperitoneally) for 7 days. The daily weights of rats inboth control and dexamethasone-treated groups were monitored. By the endof the treatment period, the mean weight of the control group hadincreased by 12±1%, whereas that of the glucocorticoid-treated group hadsharply decreased, by 16±2% (n=3 experiments, each with 3 rats pergroup). Rats were fasted for 18 h prior to each experiment and werekilled by cervical dislocation. Blood was obtained by cardiac puncturefrom anesthetized animals. The mean blood glucose concentration was5.4±0.2 mM and 10.8+0.6 mM in control and dexamethasone-treated rats,respectively, as measured with a YSI 2300STAT glucose/lactate analyser(Yellow Springs Instruments). The insulin resistant state of theskeletal muscle was further confirmed using an in vitro[¹⁴C(U)]-D-glucose incorporation assay, which demonstrated over 95%reduction in the rates of insulin-stimulated glycogen synthesis indexamethasone-treated rats (results not shown). Insulin and amylinconcentrations in the blood of normal and insulin resistant rats weredetermined as described (17). Rats with insulin resistance induced bychronic high fat feeding were generated as described previously (26).

Dissection and Metabolic Radiolabelling of Rat Skeletal Muscle Strips

Rat extensor digitorum longus (EDL) muscle strips were prepared from 18h-fasted rats. Dissection and isolation of muscles were carried outunder anaesthesia with pentobarbital (5-7 mg/100 g of body weight,intraperitoneally) as described previously (23). Each muscle was splitinto three ˜1 mm width strips. Muscle strips were pre-incubated in ashaking incubator at 30° C. for 1 h in 5 ml of Dulbecco's ModifiedEagle's medium without sodium phosphate. All incubation media weregassed with a mixture of 95% O₂ and 5% CO₂. The muscle strips weresubsequently transferred to similar flasks containing identical mediumplus 0.25 mCi/ml [³²P]-orthophosphate and incubated for a further 4 h toequilibrate the internal ATP pool (23, 24). Human insulin, rat amylin,epinephrine or CGRP were then added to the incubation media for 30 minat stated final concentrations. Reactions were terminated by freezingmuscle strips in liquid nitrogen immediately after incubation. Musclestrips were then weighed and stored at −80° C. until further analysis.

Muscle Extraction and Two-Dimensional Gel Electrophoresis (2-DE)

Muscle strips were homogenized in 2-DE lysis buffer (9M urea, 2% v/vtriton X-100, 2% v/v pharmalyte pH 3-10, 200 mM DTT, 8 mM PMSF) for 5min on ice. The lysates were briefly sonicated and microcentrifuged at12,000 rpm for 10 min to remove debris. Protein concentrations weredetermined by the Bradford method and radioactivity was measured byliquid scintillation counting. ³²P-labelled lysates with equivalentamounts of radioactivity were isoelectrically focused on IPG Drystrip pH4-7 and pH 3-10 Linear gels using a multiphor RII electrophoresis systemaccording to the manufacturer's instructions. Second dimensionalSDS-PAGE was carried out using ExcelGel™ precast 12-14% acrylamidegradient gels. After electrophoresis the gels were fixed in 10% glacialacetic acid, 40% ethanol and the proteins visualized by phosphorimagingor autoradiography. In all figures, the gels are displayed with theacidic end of the isoelectric focusing dimension to the right and thedirection of SDS-PAGE from top to bottom.

-   -   cDNA cloning, construction of expression vector and        transfection.

A full length cDNA encoding wild type rat P20 was cloned by RT-PCR,using a (SEQ ID NO: 1) forward primer5′GCCCGCGGATCCATGGAGATCCGGGTGCCTGTG3′ and (SEQ ID NO: 2) reverse primer5′GCCCGGGATCCCTACTTGGCAGCAGGTGGTGAC3′respectively. The resulting clone was validated by DNA sequencing, andthen inserted into the multiple cloning site of cytomegaloviruspromoter-driven eukaryotic expression vector pcDNA3. 1 (referred to aspcDNA.P20).

L6 myoblast cells were transfected with pCXN2-GLUT4myc (27), orco-transfected with pCXN2-GLUT4myc and pcDNA.P20, using LipofectaminePlus reagent according to the manufacturer's instructions. Stabletransfectants were selected in medium containing the neomycin analogueG418 at 400 μg/ml. At 10 days after transfection, the clones wereselected using sterilised steel rings and expanded separately in thepresence of G418. Clones that express P20 and myc-tagged GLUT4 werechosen by western blotting and used for further experiments.

Western Blotting

About 50 μg proteins from liver, heart, epididymal fat pad, aorticsmooth muscle, EDL muscle, soleus muscle tissues and whole bloodobtained from 18 h-fasted male Wistar rats were separated by SDS-PAGEand subsequently transferred to nitrocellulose membranes. The membraneswere blocked over night at 4° C. and then incubated with rabbit anti-P20polyclonal antibody (1:1000) for 2 h at room temperature. Afterincubation with streptavidin-biotinylated horseradishperoxidase-conjugated secondary antibody for another 1 h at roomtemperature, the proteins immunoreactive to the primary antibody werevisualised by enhanced chemiluminescence (ECL) detection according tothe manufacturer's instructions.

Northern Blot Analysis

Total cellular RNA was isolated from EDL muscle of 18 h-fasted controland dexamethasone-treated rats using TRIZOL reagent. 15 μg of RNA fromeach sample was separated by 1.5% agarose-formaldehyde gelelectrophoresis and subsequently transferred to Hybond-N.⁺nylonmembranes by capillary blotting in 20×SSC. The P20 cDNA probe waslabelled with ³²P-dCTP using a random primer labelling system. Themembranes were pre-incubated with hybridisation buffer (0.5 M Na₂HPO₄,pH 7.2, 10 mM EDTA, 7% SDS) for 3 h at 65° C. and subsequently incubatedwith fresh buffer containing the labelled probe for 18 h. Membranes werethen washed, analysed using a phosphorimager and quantitated by MacBASv2.5 software. For comparison, RNA samples from EDL muscle stripstreated with or without 50 nM amylin were also analysed in parallel.

Glucose Uptake Assays

L6 cells stably overexpressing myc-tagged GLUT4, or myc-tagged GLUT4plus P20, were grown in 6-well plates and differentiated into myotubesin DMEM containing 2% fetal bovine serum for 7 days. The cells weredeprived of serum for 16 h prior to experiments. For glucose uptakeassays, L6 myotubes were rinsed three times with Krebs-Henseleit buffer(KHB) and incubated in KHB with or without hormones (insulin or insulinplus amylin) at the indicated concentrations for 15 min at 37° C.Carrier-mediated glucose uptake of 10 μM 2-deoxy-D-[³H] glucose in theabove solution was measured for 15 min at 37° C. This was followed byrinsing the cells three times with ice-cold PBS and cell disruption with0.1 N NaOH. The associated radioactivity was determined by liquidscintillation counting. The protein concentration was measured with aBCA protein quantitation kit (PIERCE). The nonspecific uptake wasdetermined in the presence of 10 μM cytochalasin B and subtracted fromeach value.

Data Analysis

Autoradiography films were scanned and digitised using a Sharp JX-325scanner, and protein spots detected, quantitated and analysed using theMelanie II software package, ver. 2.2 (Biorad). The detection parameterswere: smooth 2, Laplacian threshold 3, partials threshold 1, saturation90, peakedness increase 100 and minimum perimeter 10. The matching ofmultiple features to one feature was not allowed. The pixel value is theoptical density (OD). Features were calculated as a percentage of thesum of VOL (the feature's volume, i.e., the integration of OD over thefeature's area) for all features on the gel. The radioactivity ofprotein spots was also detected by a phosphorimager and analysed byMacBAS v2.5 software. The radiation dose of each spot was displayed interms of units of photostimulated luminescence (PSL). All the resultspresented are based on at least three independent experiments.Statistical analysis was performed using the t-test (paired two sample).

Example 2 P20 is the Major Insulin Responsive Phosphoprotein in Rat EDLMuscle Detected By 2-DE

P20 was initially isolated from rat skeletal muscle as a by-productduring the purification of small heat shock proteins HSP27/28 andαB-crystallin (25). Under normal physiological conditions, it exists aslarge aggregates. P20 has been thought to be a heat-shock relatedprotein, since it has significant amino acid sequence similarity withαB-crystallin (47%) and HSP27/28 (35%) (25, 28). However, unlike othersmall HSPs, heat treatment or chemical stress does not induce theexpression of P20. Several recent studies suggest that P20 may be anactin-binding protein that is involved in cyclic nucleotide-mediatedvasodilation and relaxation of rat smooth muscle, or histamine- andphorbol ester-induced contraction of bovine carotid artery smooth muscle(29-31). Interestingly, this protein is also present at highconcentration in circulating whole blood in patients with vasculardiseases. It can strongly suppress platelet aggregation in vitro and exvivo, possibly by inhibiting receptor-mediated calcium influx inplatelets (32). However, the precise physiological functions of P20 arestill uncertain.

Analysis of the protein content of P20 by western blot showed that thisprotein is mainly expressed in rat soleus muscle, EDL muscle and heartmuscle tissues, which account for 35.1±3.2%, 29.6±2.7% and 23.3+2.5% ofthe total P20 in all the tested tissues respectively (n=3, expressed asmean±S.D.) (FIG. 1). A small amount of this protein was also detected insmooth muscle (4.9±0.6%) adipose tissue (1.9±0.3%) and blood (5.2±0.6%).2-DE analysis of ³²P-radiolabelled rat EDL muscle revealed about 150phosphoproteins labeled following insulin stimulation (FIG. 2).Quantitative analysis by Melanie II software revealed that P20 is thesecond most abundant phosphoprotein in insulin-stimulated rat EDLmuscle, representing over 2% of the total VOL for all features detected.Moreover, P20 is the only detected phosphoprotein that is responsive toboth insulin and its antagonists, as analysed by the proteome approach.

Example 3 Interplay Between Insulin and Amylin on Phosphorylation of P20

Our previous studies demonstrated that insulin and its antagonists,epinephrine, amylin and CGRP, elicit differential phosphorylation ondifferent sites of P20, thus producing three phosphorylated isoelectricvariants of P20 (termed as S1, with a pI value of 6.0; S2, with a pIvalue of 5.9; and S3, with a pI value of 5.6) (23, 24). Phosphorylationof S1 occurs at serine 157 of P20, and insulin can increase itsphosphorylation through a PI-3 kinase mediated pathway. Amylin, CGRP andepinephrine evoke phosphorylation at Ser16 of P20, through a cAMPmediated pathway, leading to the production of the phosphoisoform S2. Inaddition, these catabolic hormones also induce the phosphorylation ofP20 at another two unidentified sites to produce the phosphoisoform S3.

Here, we further investigated the interplay between insulin and severalof its antagonists on phosphorylation of P20. Interestingly, we foundthat insulin and amylin can antagonise each other's actions on thephosphorylation of this protein (FIG. 3). On the one hand,insulin-induced phosphorylation of S1 was significantly decreased in thepresence of amylin. Phosphorylation of S1 in samples treated with 50 nMinsulin plus 50 nM amylin was 49% lower than that in samples stimulatedwith 50 nM insulin alone. On the other hand, insulin blockedamylin-evoked phosphorylation of S2 and S3. In the presence of insulin,phosphorylation of S2 and S3 was decreased by about 72% and 74%respectively, relative to that in muscles treated with amylin alone.However, insulin had no effect on phosphorylation of S2 and S3 inducedby the other two catabolic hormones epinephrine and CGRP, and viceversa. This result indicates that “cross-talk” occurs only between theinsulin- and amylin-evoked signalling pathways; although all threecatabolic hormones are thought to act through G-protein coupledreceptors and to have similar metabolic effects. Amylin inhibits theinsulin-evoked PI-3 kinase cascade-mediated phosphorylation of S1.Conversely, insulin suppresses the amylin-evoked cAMP pathway-mediatedphosphorylation of S2 and S3. Such an inhibitory effect of insulin onamylin's biological actions could provide a reasonable explanation as towhy administration of exogenous amylin in physiological quantities didnot induce hyperglycemia and insulin resistance in some experimentalsystems.

The fact that insulin has separate effects on inhibition of biologicalactions of amylin and CGRP further excludes the possibility that amylinacts solely through a CGRP receptor, although the two peptide hormonesare members of the calcitonin related polypeptide family (33). Theamylin-specific receptor still remains to be identified. Several recentstudies have, however, suggested that the identity of anamylin-selective receptor may be determined in part byreceptor-activity-modifying proteins (RAMPs) (34).

Example 4 Alteration in Phosphorylation of P20, But Not Its Expression,is Associated with Insulin Resistance

We next investigated the phosphorylation patterns of P20 and the effectof insulin and amylin on this protein in dexamethasone-induced diabeticrats with insulin resistance. The diabetic state of these rats wasconfirmed by the demonstrated loss of body weight, hyperglycaemia anddecrease in insulin-stimulated incorporation of glucose into glycogen(results not shown). In dexamethasone-treated rats, both the fastedbasal plasma concentrations of insulin (789±94 pmol/l vs. 203±28 pmol/lin control rats) and amylin (144±17 pmol/l vs. 22.7±5.9 pmol/l incontrol rats) were significantly increased (p<0.01 in each case).

DL muscle strips from these rats were radiolabelled with ³²P, treatedwithout or with insulin and amylin, then phosphorylation of P20 wasanalysed by 2-DE and phosphorimaging (FIG. 4). Under the incubationconditions without hormone stimulation, phosphorylation of S2 and S3 washardly detected in the non-diabetic control rats (FIG. 4A). By contrast,these two phosphoisoforms were clearly visualised in muscle samples fromthe insulin resistant rats (FIG. 4B). Quantitative analysis byphosphorimager and MacBAS software showed that the signals associatedwith both S2 and S3 in dexamethasone-treated rats were about 5-foldhigher (Table 1). This phenomenon was also observed in a high-fatinduced insulin resistant rat model (FIG. 5), suggesting that theincreased phosphorylation of two isoforms of P20, S2 and S3, may beassociated with insulin resistant states in general. Analysis of P20expression revealed that the mRNA level and protein abundance of P20 wasnot changed either in the diabetic rats or in the amylin-treated musclestrips (FIG. 6). TABLE 1 Quantitative analysis of the radioactivityassociated with the three isoforms of P20 in non- diabetic control ratsand dexamethasone-treated rats. Non-diabetic control ratsDexamethasone-treated rats S1 S2 S3 S1 S2 S3 Basal state 434 ± 13 21.6 ±1.9 15.1 ± 2.8 439 ± 15  102 ± 6.2* 98.6 ± 4.3* Insulin 831 ± 40 20.3 ±3.4 13.3 ± 1.3 843 ± 9  96.6 ± 5.5* 92 ± 4* Amylin 191 ± 9  289 ± 20 226± 17 181 ± 11  280 ± 13 208 ± 15  Insulin + 417 ± 16 82 ± 4  6 ± 4 407 ±21  269 ± 16* 192 ± 15* AmylinRadio-labelled EDL muscle strips from control and dexamethasone-treatedrats were incubated in the absence of hormone (basal state), in thepresence of insulin (50 nM), amylin (50 nM) or both hormones.³²P-labeled isoforms of P20 (S1, S2 and S3) were separated as in FIG. 4,detected using a phosphorimager and analysed by MacBAS software. Theradioactivity of each isoform under different treatment is expressed asmean PSL values. +−. standard deviation. *indicates values that are#significantly different (P < 0.01) from corresponding values in controlrats (n '2 4).

These results indicate that the increased phosphorylation of S2 and S3is not due to the increased expression of P20, but rather to a possibledefect in the intracellular signal transduction pathways that lead togeneration of its phosphorylated isoforms.

Another major alteration in insulin resistant rats is a significantalteration of insulin's ability to inhibit amylin-evoked phosphorylationof S2 and S3. In normal rats, 50 nM insulin decreased phosphorylation ofS2 and S3 by 71.6% and 73% respectively, compared to that in samplestreated with 50 nM amylin alone (FIGS. 4E and G). In diabetic rats, onthe other hand, amylin-evoked phosphorylation of S2 and S3 was littleaffected by insulin (FIGS. 4F and H). Under this condition, theradioactivity of both S2 and S3 was around 3.3 fold higher than that ofthe non-diabetic control rats (Table 1).

Insulin resistance is a well-known effect of glucocorticoid excess, butthe mechanisms are still uncertain (35). Although muscle isquantitatively the most important tissue for glucose disposal inresponse to insulin, there are few studies on the effects ofglucocorticoids in this tissue. Administration of dexamethasone did notaffect the number or affinity of insulin receptors in skeletal musclebut reduced the insulin receptor tyrosine autophosphorylation and alsodecreased-IRS-1 activation of PI-3 kinase, suggesting the existence ofpost-receptor defects (36). It has recently been reported thatdexamethasone treatment significantly inhibited the insulin-stimulatedtranslocation of GLUT4 from an intracellular pool to the plasmamembrane, although expression of this transporter was paradoxicallyslightly increased (37).

Pieber and coworkers observed that whenever diabetes occurred indexamethasone-treated rats, the level of amylin and the ratio ofamylin/insulin (A/I), were significantly increased (38). The increase inA/I was associated with elevated content of proamylin mRNA relative toproinsulin mRNA. This study implied that amylin could also be animportant factor that contributes to the development ofdexamethasone-induced insulin resistance. The results of our presentstudy support such a role of amylin. The phosphoisoforms S2 and S3,which were hardly detected in healthy rats but could be induced byamylin, are clearly present in diabetic rats (FIG. 4B). This may be dueto the increased amylin level or A/I ratio. It is interesting to notethat, in normal rats, insulin specifically suppresses anylin's actionson phosphorylation of P20 and elevation of cAMP levels, but has nodetectable effect on the actions of two other catabolic hormones,epinephrine and CGRP (FIG. 3). Such an action of insulin wassignificantly attenuated in dexamethasone-induced diabetic rats (FIGS.4F and H). Based on these results, it is tempting to speculate that,under physiological conditions, amylin's antagonism ofinsulin-stimulated glucose disposal is inhibited by insulin itself. Theimpairment of this action of insulin may lead to the enhanced catabolicaction of amylin, and thus partly contribute to the causation of insulinresistance in dexamethasone-induced diabetic rats.

Example 5 P20 is Involved in the Regulation of Glucose Uptake Process inL6 Myotube Cells

Although the physiological role of P20 is uncertain, the high abundanceof this protein, and its diverse responsiveness to insulin and itsantagonists, suggest that it could be a mediator involved in thebiological actions of these metabolic hormones. Notably, P20 hasrecently been shown to be an actin-binding protein (31). Bothcytoskeletal actin filaments and actin-binding proteins have beensuggested to play roles in directing traffic of glucose transporters tothe cell membrane (39, 40). Interestingly, another two proteins whoseincreased expression may contribute to insulin resistance in type IIdiabetes, Rad and PED/PEA-15, are also cytoskeleton-associated proteinsinvolved in the regulation of glucose transport (41, 42). Thus it isintriguing to speculate that metabolic hormones such as insulin andamylin could regulate glucose transport by modulating thephosphorylation states of P20.

To validate this hypothesis, we have established stable transfectants ofL6 cells that overexpress P20 (FIG. 7A). Myc-tagged GLUT4 (GLUT4myc) wasalso co-expressed in these transfectants to increase insulin sensitivity(27). In the myotube cells overexpressing GLUT4myc alone, 50 nM insulinincreased 2-deoxyglucose uptake by 2.94±0.31 fold over basal level (FIG.7B). This insulin-stimulated glucose uptake was decreased by 28% in thepresence of 50 nM amylin. However, in cells overexpressing both P20 andGLUT4myc, insulin-stimulated glucose uptake was decreased significantlyby 41±3% (n=4, p<0.05), whereas the inhibitory effect of amylin wasincreased significantly by 24±2% (n=4, p<0.05). This result demonstratedthat overexpression of P20 suppresses insulin-stimulated glucose uptakeand enhances amylin's ability to inhibit insulin's action in L6myotubes, suggesting a direct role of this protein in the regulation ofglucose metabolism.

Example 6 Summary

In summary, we have-recently identified a small phosphoprotein P20 as acommon intracellular target for insulin and several of its antagonistsincluding amylin, epinephrine and calcitonin gene-related peptide(CGRP). These hormones elicit phosphorylation of P20 at its differentsites, producing three phosphorylated isoforms (S1 with pI value of 6.0,S2 with pI value of 5.9, and S3 with pI value of 5.6) (FEBS Letters 457:149-152 and 462:25-30, 1999). Here we have shown that P20 is one of themost abundant phosphoproteins in rat EDL muscle. Insulin and amylin, twohormones co-secreted from pancreatic islet β-cells; antagonise eachother's actions on phosphorylation of this protein in rat EDL muscle.Insulin inhibited amylin-evoked phosphorylation of S2 and S3, whileamylin decreased insulin-induced phosphorylation of S1. In rats madeinsulin resistant by dexamethasone treatment, the phospho-isoforms S2and S3, which were barely detected in healthy rats in the absence ofhormone stimulation, were significantly increased. Moreover, the abilityof insulin to inhibit amylin-evoked phosphorylation of these twoisoforms was greatly attenuated. These results suggest that alterationsin phosphorylation of P20 could contribute to the development of insulinresistance.

Throughout this application, various publications are referred to bypartial citations within parenthesis. Full citations for thesepublications may be found at the end of the specification. Thedisclosures of these publications, in their entireties, are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

DOCUMENTS

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1. A method for diagnosing insulin resistance in an individualcomprising obtaining a biological sample from the individual anddetermining a level of at least one of P20 isoforms S2 and S3, whereinthe individual is diagnosed as being insulin resistant when the level ofexpression of at least one of S2 and S3 is higher than a reference levelcharacteristic of an individual not suffering from insulin resistance.2. The method according to claim 1 wherein the cells in the biologicalsample are contacted with insulin ex vivo.
 3. The method according toclaim 1 wherein the levels of both S2 and S3 are determined.
 4. Themethod according to claim 3 wherein the levels of both S2 and S3 arehigher than a reference level characteristic of an individual notsuffering from insulin resistance.
 5. A method for diagnosing insulinresistance or a propensity to insulin resistance in an individualcomprising determining the level of expression of at least one of P20isoforms S2 and S3 in a cell of an individual, and comparing the levelto a reference level characteristic of a cell of the same type in anindividual not suffering from insulin resistance or diabetes wherein alevel of expression that is higher than the reference level isdiagnostic of insulin resistance or a propensity to insulin resistancein the individual.
 6. The method of claim 5 wherein the levels of bothS2 and S3 are determined.
 7. The method of claim 6 wherein the levels ofboth S2 and S3 are higher than the reference level.
 8. The method ofclaim 5 wherein the level of expression of S2 and/or S3 is the same asgreater than a second reference level, wherein said second referencelevel is characteristic of an individual with insulin resistance.
 9. Amethod of assessing the efficacy of a treatment for insulin resistancein an individual comprising monitoring the level of at least one of S2and S3 in the individual to whom the treatment has been administered.10. A method of treating insulin resistance in an individual comprisingadministering a treatment or an agent that reduces the level of P20isoforms S2 and S3 in the individual.
 11. The method according to claim10 wherein the agent is identified by a method for screening for anagent useful for treatment of insulin resistance comprising contacting amammalian cell expressing P20 and the agent and determining if the agentsuppresses the level of at least one of P20 isoforms S2 and S3, whereinthe suppression of S2 and S3 levels is indicative of an agent useful fortreatment of insulin resistance.